Need denature (94oc in 5 mintute) for agarose gel? - (May/01/2007 )

I am a bit confuse about this issue: does it need to denature PCR products (94oC in 5 minutes) before loading on 4% agarose gel or not?. Denaturation is needed for PAGE gel for single strand DNA, but do not know about agarose gel.

-quangtranho-

Is this just for standard agarose gel electrophoresis of PCR products?? if so there is no need to denature before loading, and also your agarose concentration seems awfully high. I only ever use a 1% gel for running my PCR products.

-lauralee-

Yes, agree with lauralee that the percentage of your agarose gel is too high. The percentage of your agarose depends on the size of the product you wish to separate.

-virus_fan-

QUOTE (virus_fan @ May 4 2007, 08:18 AM)

Yes, agree with lauralee that the percentage of your agarose gel is too high. The percentage of your agarose depends on the size of the product you wish to separate.

which then follows, what is the DNA band size that you are trying to resolve? If you band size is very small, PAGE would probably be better at resolving your band.

-perneseblue-

on 3%gel, i had a nice 50bp ladder well separated. So in 4% what are the length you need to discriminate ?For denaturation, you may add you loading buffer and heat the whole at 65° 5' loading directly (whithout spin as your DNA is "heavy" molecule that does not goes in vapor).Gave me nice results with an hairpin.Btw, i don't denaturate my pcr products when loading on 3%gel. Just load them. If 1st result is not that clear, i try to denature.

-fred_33-

Well, if the product is too small, isnt it better to use PAGE gel instead for better resolution?And also use TBE instead of TAE.

-timjim-

Well, the PCR products size about 70 and 80 bp, so by separating at 4% agarose I use 20 bp ladder (ready to use from Fermenats).

I agree that PAGE give better results, but it takes time to prepare and also staining.

-quangtranho-

Well, at least you will get a better resolution and sensitivity using PAGE.